Fig. 1: Illustration of the strategy for semi-rational engineering of protein dynamics.

Exploratory phase—1 A thermostable ancestral protein, Anc^HLD-RLuc, provided a robust and evolvable template that can withstand the destabilizing effects of protein backbone engineering. 2 Libraries of single triplet insertion and deletion variants were created following the TRIAD method based on the use of engineered transposons (TransIns and TransDel)^12,13. 3 Screening of the libraries led to identification of the improved insertion mutant AncINS. 4 Twenty-five mutants with significant changes in luciferase (LUC) and haloalkane dehalogenase (HLD) activities were characterized using bioinformatics, microscale techniques (nano differential scanning fluorimetry—nanoDSF), and microfluidics. 5 Structure-function relationships were described employing partial least squares (PLS) multivariate statistics. 6 Dynamic elements required for efficient catalysis were identified by structural, kinetic, biophysical and computational characterization (molecular dynamics (MD) simulations) of AncINS. Validation phase—7 Knowledge obtained during the exploratory phase was validated by transplanting a relevant dynamic fragment from the specialized descendant into the ancestor, yielding an enzyme, AncFT, with 7000-fold higher catalytic efficiency than Anc^HLD-RLuc and 100-fold longer glow-type bioluminescence than the flash-type Renilla luciferase RLuc8. Key mutants discussed in this study are highlighted in yellow.